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SAM 903.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 903

Supplemental Assay Method for Testing for Preservative Interference with 

Sterility Tests

Date: January 3, 2022

Number: SAM 903.05

Supersedes: SAM 903.04, December 18, 2015

Standard Requirement: 9 CFR 113.25

Contact: Sophia G. Campbell, (515) 337-7489

Approvals: /s/Melisse M. Schilling Date: 01/20/2022

Melisse M. Schilling, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 01/20/2022

Paul J. Hauer, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents

4. Performance of the Test

4.1 Broth media

4.2 Agar media

5. Interpretation of the Test Results

6. Record and Report of Test Results

7. References

8. Summary of Revisions

Appendices
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1. Introduction

This Supplemental Assay Method (SAM) describes the test procedure used to determine the ratio 

of inoculum to medium for sterility testing that will result in a sufficient dilution of the product 

to prevent bacteriostatic and fungistatic activity, per title 9, Code of Federal Regulations 

(9 CFR), part 113.25(d).

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 30o- 35oC incubator 

2.1.2 20o- 25oC incubator 

2.1.3 Laminar-flow Class II biosafety cabinet (BSC)

2.1.4 Waterbath

2.2 Reagents/supplies

Equivalent reagents or supplies may be substitued for any brand name listed below.

2.2.1 Bacillus subtilis (American Type Culture Collection (ATCC) #6633) or 

equivalent organism as specified in the current United States Pharmacopoeia 

(USP)

2.2.2 Issatchenkia orientalis (ATCC #6258) or equivalent organism as 

specified in the current USP

2.2.3 Clostridium chauvoei spores or equivalent organism as specified in the 

current USP

2.2.4 Brain-Heart Infusion Agar (BHIA), National Centers for Animal Health 

(NCAH) Media #10204 (Appendix I) 

2.2.5 Trypticase Soy Agar (TSA) or Soybean-Casein Digest Agar (SCDA), 

NCAH Media #10487 (Appendix II)

2.2.6 Soybean Casein Digest Medium (SCDM) or Trypticase Soy Broth (TSB)

NCAH Media #10423 (Appendix III) 
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2.2.7 Fluid Thioglycollate Medium (FTM), NCAH Media #10135 (Appendix 

IV) 

2.2.8 Fluid Thioglycollate Medium with Beef (FTM w/Bf), NCAH Media 

#10227 (Appendix V) 

2.2.9 Sterile pipettes, individually packaged

2.2.10 Lab coat or sterile sleeves and gloves

 

2.2.11 70% ethanol

2.2.12 4 x 4-inch sterile gauze pads

2.2.13 Sterile syringes with needles

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques and preparation, proper handling, and disposal of 

biological agents, reagents, tissue culture samples, and chemicals. Personnel must also 

have knowledge of safe operating procedures and policies, as well as training in the 

operation of the necessary laboratory equipment listed in Section 2.1.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Turn on the BSC at least one hour prior to testing.

3.2.3 Monitor the temperature of incubators, freezers, and coolers according to 

SOPs.

3.3 Preparation of reagents

3.3.1 B. subtilis stock culture is prepared according to the manufacturer’s 

instructions and titrated to determine colony forming unit (CFU) concentration.

3.3.2 I. orientalis stock culture is prepared according to the manufacturer’s 

instructions and titrated to CFU concentration.
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3.3.3 C. chauvoei stock culture is prepared according to the manufacturer’s 

instructions and titrated to determine the proper use-dilution.

3.3.4 To test for preservative interference of aerobic bacterins and killed virus 

biologics, use B. subtilis with FTM at 30o- 35oC and I. orientalis with TSB or

FTM at 20o- 25oC. For anaerobic bacterins, use C. chauvoei with FTM w/Bf at 

30o-35oC and I. orientalis with TSB or FTM at 20o- 25oC. For parenteral live viral 

products, use B. subtilis with TSB at 30o- 35oC and I. orientalis with TSB at 

20o- 25oC. For chicken embryo origin (CEO) and certain poultry vaccines used for 

mass inoculation via drinking water, use B. subtilis with BHIA at 30o- 35oC and I.

orientalis with BHIA at 20o- 25oC.

4. Performance of the Test 

4.1 Broth media

4.1.1 For each biological product being tested, prepare 10 test vessels of each 

media specified for both incubation temperatures. In addition to the media for 

each test sample, prepare 10 test vessels of the appropriate media for each 

indicator organism to serve as positive controls (Section 3.3.4).

4.1.2 For each sample being tested, inoculate each of the 20 prepared vessels 

with 1 mL or 0.2 mL of the sample; the volume of inoculum depends on the 

requirement of the sterility test (9 CFR 113.26 or 113.27). 

4.1.3 Following inoculation with the test sample(s), add approximately 100 

CFU of the appropriate indicator organism to the sample test vessels. Swirl the 

vessels to distribute the sample and the organism in the medium.

4.1.4 Inoculate each of the 20 prepared vessels for the positive controls with 

approximately 100 CFU of the appropriate indicator organism. Swirl the vessels

to distribute the organism in the medium.

4.1.5 Incubate and observe for growth all test vessels inoculated with B. subtilis

or C. chauvoei at 30o- 35oC for 7 days and test vessels inoculated with I. orientalis

at 20o- 25oC for 14 days.

4.2 Agar media

4.2.1 Agar pour-plates are used for sterility testing CEO and other poultry 

vaccines administered via drinking water. For each biological product being 

tested, prepare 10 sterile petri dishes for each incubation temperature. In addition 

to the the test sample, prepare 10 petri dishes for each indicator organism (Section 

3.3.4).
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4.2.2 For each test sample, inoculate all 20 petri dishes with 10 bird doses of 

product. Following distribution of the test sample, add approximately 100 CFU of 

the appropriate indicator organism to 10 sample petri dishes. Following 

preparation of the test sample petri dishes, distribute approximately 100 CFU of 

of indicator organism into 10 petri dishes each.

4.2.3 Following distribution of the test sample(s) and indicator organisms into

the petri dishes, gently pour the appropriate volume of melted BHIA (cooled to

about 50o-60°C) into each petri dish. Gently swirl the petri dishes to mix the 

inoculum into the agar. Allow the agar to cool and harden for up to 2 hours before 

inverting for incubation.

4.2.4 Incubate the plates inoculated with B. subtilis at 30o- 35oC for 7 days and 

the plates inoculated with I. orientalis at 20o- 25oC for 14 days.

5. Interpretation of the Test Results

5.1 There is no interference in the broth test if the vessels containing biological 

product exhibit equivalent growth of the indicator organism compared to the positive 

control vessels containing no biological product.

5.2 There is no interference in the agar test if the plates containing biological product

have an average CFU within 20% of the positive control plates containing no biological

product.

5.3 If interference is demonstrated by reduced growth of the indicator organisms in 

the presence of the biological product, the test procedure must be repeated using a range 

of greater media volumes to establish the appropriate volume of media required for 

sterilty testing.

6. Record and Report of Test Results

Record and report results of the test(s) according to SOPs.

7. References 

7.1 Title 9, Code of Federal Regulations, part 113.25(d), U.S. Government Printing 

Office, Washington, DC.

7.2 The U.S. Pharmacopoeia, 1985, Vol. 21, pp 1151-1160, Mack Publishing Co., 

Easton, PA.
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7.3 Kurtzman, C. P., C. J. Robnett, and E. Basehoar-Powers. 2008. Phylogenetic 

relationships among species of Pichia, Issatchenkia and Williopsis determined from 

multigene sequence analysis, and the proposal of Barnettozyma genera novel, Lindnera 

genera novel and Wickerhamomyces genera novel. FEMS Yeast Res 8:939-54.

8. Summary of Revisions

Version .05

• The Bacteriology Section Leader has been updated.

Version .04

• The Bacteriology Section Leader has been updated.

• Updated Sections 1, 2, and 4.1.

• Appendices: Updated media storage limits to be in compliance with 9 CFR 

113.25(b).

Version .03

• Sections 1-4 have been updated to reflect current practices.

• 7.3: Reference added for name change of Candida krusei to Issatchenkia orientalis.

Version .02

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

• The document number has been changed from STSAM0903 to SAM 903.

• The Contact has been changed from Gerald Christanson to Sophia Campbell.

• 2.1: The equipment/instrumentation list has been updated to include sterile test 

vessels and to clarify that the Biosafety Cabinet is to be Class II.

• Appendices: Media storage conditions have been added.
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Appendices

Appendix I

NCAH Media #10204

Brain Heart Infusion Agar (BHIA)

Brain Heart Infusion Agar 52.0 g

QH2O 1000 mL

Autoclave 20 minutes at 121oC. Store at 2°- 5°C for up to 90 days.

Appendix II

NCAH Media #10487

Trypticase Soy Agar (TSA) or Soybean-Casein Digest Agar (SCDA)

Trypticase Soy Agar 40.0 g

QH2O 1000 mL

Autoclave 20 minutes at 121oC. Store at 2°- 5°C for up to 90 days.

Appendix III

NCAH Media #10423 

Trypticase Soy Broth (TSB) or Soybean-Casein Digest Medium (SCDM)

Trypticase Soy Broth 30.0 g

QH2O 1000 mL

Autoclave 20 minutes at 121oC. Store at 20°- 25°C for up to 90 days.

TSB and SCDM are synonymous mediums. 
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Appendix IV

NCAH Media #10135 

Fluid Thioglycollate Medium (BBL)

Fluid Thioglycollate Medium 29.5 g

QH2O 1000 mL

Mix and heat to boiling. Autoclave 20 minutes at 121°C. Store at 20°- 25°C for 

up to 90 days.

Appendix V

NCAH Media #10227

Fluid Thioglycollate with Beef Extract

Fluid Thioglycollate Medium 29.5 g

QH2O 1000 mL

Heat and add:

0.5% Beef Extract (Difco) 5.0 g

Bring to a boil and dispense. Autoclave 20 minutes at 121oC. Store at 20°- 25°C 

for up to 90 days.